Difference between revisions of "Part:BBa S03595:Design"

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Once the '''Double Forward Terminator''' [https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015] is placed upstream of RBS-TetR, the cells are no longer tetracycline resistant. We conclude that the '''Double Forward Terminator''' is sufficient to block read-through transcription.
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Once the '''Double Forward Terminator''' [https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015] is placed upstream of RBS-TetR, the cells are no longer tetracycline resistant. We conclude that the '''Double Forward Terminator''' is sufficient to block read-through transcription. Thus, the '''Double Forward Terminator''' was used to contruct a new cloning vector '''pSB1A4''' ([https://parts.igem.org/wiki/index.php/Part:BBa_J31009 BBa_J31009]).
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Construction of an "insulator vector" where the double terminator is placed before the BioBrick prefix and after the BioBrick suffix (in the reverse orientation) is currently under way. This vector should be able to insulate devices from forward and reverse read-through allowing total control over expression within the device.
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===Source===
 
===Source===

Revision as of 17:07, 19 October 2006


TT : RBS-TetF


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 302
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 448
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 474
    Illegal NgoMIV site found at 842
    Illegal NgoMIV site found at 1002
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Read-through transcription is extremely problematic when the usefulness of a device depends upon an off state. This part was constructed to test the capacity of the Double Forward Terminator BBa_B0015 to insulate a coding region from read-through transcription.


Part BBa_S03562, which contains a tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF), is sufficient to convey tetracycline resistance in the absence of a promoter. We suspect that Tet might be expressed via forward read-through transcription from the carrier vector (pSB1A2 and pSB1A3). Consistent with this observation, the following parts, which are predicted to not show expression, do show expression

  • BBa_S03532 - contains a tetracycline resistance coding region (backward orientation) with a backwards ribosomal binding site (TetB-RBS)
  • BBa_J3106 - contains the pBad promoter in the forward orientation followed by TetB-RBS in the reverse orientation (pBad-hixC-TetB-RBS-hixC-TT-RE)
  • BBa_J44004 - contains the pBad promoter in the reverse orientation followed by RBS-TetF (hixC-pBadrev-hixC-RBS-TetF)
  • RFP (red flourescent protein) - contains RBS-RFP with no promoter

Note that backwards Tet is also expressed without a promoter. This suggests that there is also reverse read-through from the carrier vector.


Once the Double Forward Terminator BBa_B0015 is placed upstream of RBS-TetR, the cells are no longer tetracycline resistant. We conclude that the Double Forward Terminator is sufficient to block read-through transcription. Thus, the Double Forward Terminator was used to contruct a new cloning vector pSB1A4 (BBa_J31009).

Source

References